The ubiquitin-proteasome pathway is responsible for the majority of intracellular protein degradation, and plays an essential role in fundamental cellular processes such as mitosis and apoptosis. Our past efforts helped establish the proteasome as a therapeutic target by showing that inhibitors induce preferential apoptosis in c-myc-transformed cells, demonstrating their activity in in vivo models, and documenting their safety and efficacy in Phase I trials. These studies led to Phase II multicenter trials that confirmed the activity of one such inhibitor, bortezomib, which has now been approved by the FDA for clinical use. While these inhibitors likely trigger apoptosis through several pathways, our recent work implicates an important role for inhibition of p44/42 MAPK by induction of MKP phosphatases. Preliminary evidence suggests MKP-1 induction occurs in part through p38 MAPK, and that MKP-1 may also be anti-apoptotic by decreasing JNK activity, since p38 inhibitors decrease MKP expression, and enhance apoptosis and phospho-JNK levels. To expand upon these findings, we propose to: 1. Study the role of p38 MAPK, and of MKP-1 in proteasome inhibitor-mediated apoptosis. Pharmacologic p38 inhibitors will be used in conjunction with mutant p38 and MKP-1 constructs, p38- and MKP-knockout cells, as well as xenografts, to test the hypotheses that p38 activation and MKP expression are important anti-apoptotic elements; 2. Evaluate the involvement of the downstream p44/42 targets p90RSK and Bad. Pharmacologic MEK inhibitors, as well as mutant p90RSK and Bad constructs, will be used to test the hypotheses that p90 and Bad are major pro-apoptotic targets of proteasome inhibitors, and that p44/42 pathway inhibition enhances apoptosis; and 3. Determine the potential of dual MAPK blockade with proteasome inhibition. Since p38 and MKP inhibition enhances p44/42 activity, which we have shown is anti-apoptotic, we suspect that blockade of both pathways together should further enhance the anti-tumor efficacy of proteasome inhibition. Taken together, these studies will further clarify some of the mechanisms by which proteasome inhibitors induce apoptosis, identify agents that may increase their efficacy, and evaluate these regimens with in vivo models. Since p38 and MEK inhibitors are currently undergoing clinical development, this work will establish the framework for translation of novel, rational proteasome inhibitor-based combination regimens with the potential for enhanced anti-tumor efficacy into the clinical arena.